POTASSIUM CHANNELS SUPPORT ANION SECRETION IN PORCINE VAS DEFERENSEPITHELIAL CELLS

by

PRADEEP REDDY MALREDDY

B.V.Sc & A.H.College of Veterinary Science,

Acharya N.G Ranga Agricultural University.2001

A THESIS

submitted in partial fulfillment of the requirements for the degree

MASTER OF SCIENCE

Department of Anatomy and PhysiologyCollege of Veterinary Medicine

KANSAS STATE UNIVERSITYManhattan, Kansas

2009

Approved by:

Major Professor

Bruce D. Schultz

Abstract

Epithelial cells lining the vas deferens modify the luminal contents to which sperm are

exposed in response to neuroendocrine, autocrine and lumicrine transmitters. The role and

identity of vas deferens epithelial potassium channels that provide the correct luminal

environment for sperm maturation and delivery have not yet been determined. Cultures of vas

deferens epithelial cells isolated from adult pigs were employed to investigate contributions of

selected ion channels to net flux. A two-pore potassium channel, TASK-2, was identified on the

apical membrane of cultured primary porcine vas deferens epithelial cells (1°PVD). Bupivacaine,

a known TASK-2 inhibitor, when added to the apical bathing solution, inhibited forskolin-

stimulated short circuit current, Isc, in a concentration dependent manner with a maximum

inhibition of 72 ± 6% and an IC50 of 7.4 ± 2.2 µM. Apical exposure of 1°PVD cells to quinidine,

lidocaine, and clofilium (other known TASK-2 blockers) inhibited forskolin-stimulated Isc in a

concentration dependent manner. Fitting a modified Michalis-Menten function to the data

revealed IC50 values of 274 µM, 531 µM, and 925 µM, respectively. Riluzole, a two-pore

potassium channel activator, stimulated bupivacaine-sensitive Isc, further confirming the

contribution of TASK-2 to net ion flux. Western blotting demonstrated the presence of TASK-2

immunoreactivity in 1°PVD cell lysates, while immunocytochemistry demonstrated apical

localization of the targeted epitope in virtually all cells lining native porcine vas deferens. These

results suggest that TASK-2 likely plays a role in vas deferens epithelial ion transport that may

account for the reportedly high concentration of potassium in the male reproductive duct lumen.

TASK-2 likely contributes to male fertility as an integral member of the regulated transport

processes that account for the luminal environment to which sperm are exposed.

iii

Table of Contents

List of Figures.............................................................................................................................v

List of Tables.............................................................................................................................vi

Acknowledgements...................................................................................................................vii

Dedication ...............................................................................................................................viii

CHAPTER 1 - Introduction.........................................................................................................1

1.1 Overview of K+ Channels..................................................................................................1

1.2 TASK Channels ................................................................................................................4

1.3 Identification of TASK-2 K+ Channels ..............................................................................5

1.4 References ........................................................................................................................7

CHAPTER 2 - TASK-2 potassium channel supports ion secretion in pig vas deferens epithelia 12

2.1 Abstract...........................................................................................................................13

2.2 Introduction.....................................................................................................................14

2.3 Materials and Methods ....................................................................................................16

2.3.1 Culture of Porcine Vas Deferens Epithelial Cells (1°PVD Cells)...........................16

2.3.2 Electrophysiology.................................................................................................17

2.3.3 Immunohistochemistry .........................................................................................18

2.3.4 1°PVD Whole Cell Lysate Preparation .................................................................18

2.3.5 Pig Kidney Cortex Lysate Preparation ..................................................................19

2.3.6 Western Blotting...................................................................................................20

2.3.7 Chemicals.............................................................................................................20

2.3.8 Data Analysis .......................................................................................................21

2.4 Results ............................................................................................................................21

2.4.1 Apical Bupivacaine Inhibits Forskolin-Stimulated Isc in 1°PVD Cells ...................21

2.4.2 Lidocaine, Clofilium and Quinidine Inhibit Forskolin-Stimulated Isc across 1°PVD

Monolayers ...................................................................................................................23

2.4.3 Riluzole, a two-pore potassium channel activator stimulates Isc across 1°PVD cells

......................................................................................................................................27

2.4.4 1°PVD cells express TASK-2 immunoreactivity...................................................28

iv

2.4.5 Apical localization of TASK-2 protein in native porcine vas deferens ducts..........29

2.5 Discussion.......................................................................................................................30

2.6 Acknowledgements .........................................................................................................37

2.7 References ......................................................................................................................37

CHAPTER 3 - Potential experiments and future directions .......................................................45

3.1 Overview ........................................................................................................................45

3.2 References ......................................................................................................................50

v

List of Figures

Figure 1.1 Schematic diagram describing K+ channel basic features............................................1

Figure 1.2 Topology of K+ channel α-subunits............................................................................3

Figure 2.1 Forskolin-stimulated Isc in primary porcine vas deferens epithelial cell monolayers

(1°PVD cells) is inhibited by apical application of bupivacaine. ..............................23

Figure 2.2 Forskolin-stimulated Isc across 1°PVD cells is inhibited by apical exposure to

lidocaine, clofilium or quinidine. .............................................................................26

Figure 2.3 Apical application of riluzole stimulates Isc across 1°PVD cells................................28

Figure 2.4 TASK-2 immunoreactivity is present in 1°PVD cell lysates. ....................................29

Figure 2.5 TASK-2 immunoreactivity is present in cells lining native porcine vas deferens.......30

Figure 2.6 Porcine vas deferens epithelial cell model for ion transport. .....................................35

vi

List of Tables

Table 1.1 Candidate genes for K+ conductance in human vas deferens epithelial cells. ................7

vii

Acknowledgements

I thank my advisor and mentor Dr. Bruce D Schultz for all the guidance and support he

has given me over last many years. He has helped me with some tough decisions for my career. I

shall be very grateful for that. I extend my sincere gratitude to Mr. Ryan Carlin who taught me

everything from the very basics of making solutions to conducting Ussing chamber experiments.

I thank my fellow graduate students Drs. Rebecca Quesnell and Suma Somasekharan for their

helpful nature and constructive criticism of my research endeavors. I thank Terry O’Leary for

teaching me the basics of PCR. I thank Dr. Fernando Alves for having helpful discussions on

almost anything related to work and life. I thank my fellow lab members Florence Wang, Dr.

Vladimir Akoyev, Cameron Duncan, Elizabeth Blaesi and Qian Wang for creating pleasant

work atmosphere. I thank Dr. Daniel Marcus for teaching me some of the basics of

bioelectricity, letting me use the equipment in his laboratory and serving on my supervisory

committee. I thank Dr. Philine Wangemann and Joel Sanneman for their help with confocal

microscopy. I thank my other committee members, Dr. Lisa Freeman and Dr. Larry

Takemoto for all their help. I am most thankful to Drs. Walter Cash, Judy Klimek, Deryl

Troyer and Jim Polikowski for their help with teaching Gross Anatomy to freshmen DVM

students. I thank Dani, Bonnie, and Jenny for their administrative support. I thank my friends

Dr. Bala Thiagarajan, Jessica Eichmiller, Nithya Raveendran, Dr. Satya Pondugula, Dr.

Satish Medicetty and Dr. Raja Rachakatla for their support and guidance. I thank all my

friends here at K-State, Sairam Jabba, Chanran Ganta, Kamesh, Kiran, Praveen, and Shiva.

viii

Dedication

I dedicate this work to the late Dr. Prakash Krishnaswami and his wife Sujatha

Prakash and my family.

1

CHAPTER 1 - Introduction

1.1 Overview of K+ Channels

Potassium channels are the most diverse group of proteins that are encoded by at least

100 different genes in humans alone (Hietzmann et al, 2008)(30). Potassium channels are

expressed in almost all cell types including both excitable and non-excitable cells. All potassium

channels share three basic properties: they have high conduction rates of about 107 to 108 ions

per second, are selective for potassium ions and their conduction is gated (Doyle et al, 1998)

(Fig. 1.1).

Figure 1.1 Schematic diagram describing K+ channel basic features.

M1 and M2 represent transmembrane domains. The purple and green balls represent the relative

dehydrated sizes of K+ and Na+ respectively. The K+ is almost twice the size of Na+. Yet Na+, which is

smaller, is essentially excluded as indicated by the green ball turning away from the pore. The conduction

is turned on and off by opening and closing a gate, which can be regulated by an external stimulus such as

ligand-binding or membrane voltage. (Fig. modified from Doyle et al, 1998).

2

The potassium channels present in non-excitable cells (e.g., epithelia) can be categorized

broadly into three different groups based on structure: The first group is characterized by six

transmembrane domains and one P-loop that is part of the ion-conducting pore (6TMD/1P, Fig.

1.2A), which includes voltage-dependent (Kv) and calcium-activated (KCa) K+ channels. Kv

channels are gated by membrane voltage, i.e., they open and close in response to changes in

membrane potential. Kv channels comprise a large group of K+ channels encoded by at least 40

different genes in humans (Gutman et al, 2003 and 2005) and are preferentially expressed in

excitable cells. Kv channels expressed in non-excitable cell types such as renal (Hebert et al,

2003), gastro-intestinal (Hietzmann et al, 2008) and respiratory epithelia (Bardou et al, 2009)

have an essential role in various functions including electrolyte and substrate transport, cell

volume regulation, cell migration, wound healing, proliferation, apoptosis, carcinogenesis, and

oxygen sensing (O’Grady et al, 2005). KCa channels are gated by intracellular Ca2+

concentration. Ca2+ may interact directly with the channel protein or may cause a secondary

signaling cascade that may then either activate or inhibit the channel. KCa channels have been

studied extensively in the nervous system where they play an important role in regulation of

intracellular Ca2+ concentration. KCa in epithelial cell types have been shown to play an

important role in O2 sensing in alveoli (Jovanovic et al, 2003), kidney cell migration (Schwab et

al, 2001), inflammatory responses in lung pathologies (Dulong et al, 2007) and control of Cl-

secretion in airways (Devor et al, 2000; Cowley et al, 2002; Bernard et al, 2003)

The second group is characterized by two TMD and one P-loop (2TMD/1P, Fig. 1.2C),

which includes inward-rectifier K+ channels (Kir) (Bardou et al, 2009). A functional Kir channel

like Kv channels, is believed to be a tetramer. However, a striking biophysical property of these

3

channels is the inward rectification, which refers to greater K+ conductance under

hyperpolarization and lesser conductance under depolarization, the effect opposite to that seen in

Kv channels (Lu, 2004). These K+ channels are involved in repolarization of action potentials,

setting the resting potential of the cell and contributing to K+ homeostasis. Gene mutations of

inward rectifiers have been linked to several human diseases including Bartter syndrome (Simon

et al, 1996) and Andersen’s syndrome (Plaster et al, 2001).

Figure 1.2 Topology of K+ channel α-subunits.

The numbers (1-6) represent the transmembrane segments. A, B, C) Each panel represents an α-subunit

of a well-described class of K+ channels. A, C) A functional K+ channel is formed by the homo- or hetero-

tetramerization of subunits. B) A functional K2P channel is formed by the dimerization of 4TMD/2P sub-

units. (Modified from Bardou et al, 2009).

The third group is characterized by four TMD and two P-loops (4TMD/2P, Fig. 1.2B),

which includes two-pore K+ channels (K2P) (Lesage et al, 2000). The first member of the two-

pore K+ channel family was named TWIK-1 for Tandem of P domains in a Weak Inward

Rectifying K+ channel. Currently, fifteen mammalian genes have been identified that code for

K2P family members. K2P channels are further classified in to six subfamilies based on their

functional domains and include TWIK, TREK, TASK, TALK, THIK and TRESK. K2P K+

4

channels are expressed in a number of different cell types and have distinct functions depending

on the channel subtype expressed.

1.2 TASK Channels

TWIK related Acid Sensitive K+ (TASK) channels are expressed in many tissues and cell

types including the brain, heart, lung, kidney, small intestine, liver, pancreas, placenta and testis

(Reyes et al, 1998). TASK channels are constitutively active near resting membrane potential

(Duprat et al, 1997) and thus were also referred to as background or leak channels. The TASK

channel family is comprised of five different isoforms: TASK-1, TASK-2, TASK-3, TASK-4,

and TASK-5. All these isoforms are sensitive to extracellular pH, which makes them strong

candidates for the K+ conductance(s) contributing to, or regulating pH-sensitive ion transport

systems. For example in murine kidney proximal tubules, TASK-2 channels are activated by an

increase in luminal pH due to bicarbonate transport (Warth et al, 2004). TASK-2 is also involved

in the regulation of apoptosis in kidney proximal tubule cells. Apoptosis in TASK-2 knock out

mice was significantly reduced suggesting a role in apoptotic volume decrease in kidney

proximal tubule cells (L’Hoste et al, 2007a). TASK channels are known to be involved in

neuronal protection (Liu et al, 2005) and general anesthesia (Patel et al, 1999). Expression of

TASK channels in hippocampal cells resulted in a significant protection from an oxygen-glucose

deprivation injury and this neuroprotection was enhanced in the presence of isoflurane, a general

anesthetic (Liu et al 2005). Involvement of TASK-2 has been suggested in volume regulation of

spermatozoa in mice (Barfield et al, 2005). It is known that spermatozoa have high K+

concentration (Chou et al, 1989) and that K+ concentration increases in luminal fluids along the

5

length of epididymis and vas deferens (Turner et al, 2002). Hence a role for TASK-2 in the vas

deferens is plausible.

1.3 Identification of TASK-2 K+ Channels

Pharmacological agents that selectively either block or activate an ion channel have been

employed to implicate contributions of distinct channels to overall tissue functions. Of the wide

range of K+ channel blockers available, tetraethyl ammonium (TEA), 4-aminopyridine (4-AP),

Cs+, and Ba2+ have been used as broad-spectrum K+ channel blockers. However, K2P channels

are relatively insensitive to these K+ channel blockers.

Local anesthetics including bupivacaine and lidocaine inhibit TASK-2 in heterologously

expressed cells in a concentration dependent manner. These local anesthetics are also known to

inhibit other K2P channels including TASK-1 and TASK-3 and voltage-gated Na+ channels. The

concentration required to inhibit conductance by 50% (IC50) for TASK-2 inhibition by

bupivacaine in HEK293 cells is in the low to mid-micro molar range (17 µM; Kindler et al,

2003), and the IC50 for inhibition of voltage-gated Na+ channels in HEK293 cells is in the same

range (4.5 µM; Wang et al, 2001). The effects of quinine and quinidine on currents elicited by

voltage pulses to +50 mV have been studied in TASK-2-expressing COS cells. Quinidine, at 100

µM, induced a 65 ± 3.8% inhibition of the current in these cells (Reyes et al, 1999). It has been

reported that clofilium inhibited K+ currents in mTASK-2-transfected HEK293 cells in a

concentration dependent manner (Niemeyer et al, 2001) with an IC50 of ~25 µM. Thus

bupivacaine, lidocaine, quinidine, and clofilium can provide pharmacological profile to

characterize TASK-2 K+ channel currents.

6

Riluzole reportedly activates TASK-2 channels in Calu-3 cells, a cell line derived from

human airway (11), an attribute that makes it potentially valuable for the proposed studies.

However this compound has been shown also to activate TREK-1 and TRAAK, two members of

K2P family that are closely related to TASK-2 and riluzole has been shown to affect other classes

of ion channels. For example riluzole was shown to activate small conductance, Ca2+-activated

K+ channels (SK channels) expressed HEK293 cells (Cao et al, 2002). These activities of

riluzole across classes of ion channels greatly diminish the diagnostic value of the drug.

Nonetheless, riluzole can be used as one component in a pharmacological toolbox that is

assembled to either implicate or exclude a contribution of TASK-2 to the physiological function

of a tissue.

The selection of K2P channels in general and TASK-2 channels specifically as a focus

point for the proposed studies was built on a limited amount of preliminary information.

Specifically, microarray analysis of RNA isolated from human vas deferens epithelial cells

revealed message coding for various K+ channels including K2P channels (Table 1). Ultimately,

experiments were conducted employing cells and tissues isolated from pigs because of their

availability. Outcomes of initial experiments guided the research to focus on TASK-2.

7

Table 1.1 Candidate genes for K+ conductance in human vas deferens epithelial cells.

KCa Kv Kir K2P

KCNN4 (SK4) KCNC4 (Kv3.4) KCNJ15 (Kir1.3) KCNK1 (TWIK1)

KCNMB4 (MaxiK) KCND1 (Kv4.1) KCNJ2 (Kir2.1) KCNK6 (TWIK2)

KCNQ1 (Kv7.1) KCNJ16 (Kir5.1) KCNK3 (TASK1)

KCNQ3 (Kv7.3) KCNJ3 (GIRK) KCNK5 (TASK2)

KCNS1 (Kv9.1) KCNK17 (TASK4)

KCNS3 (Kv9.3) KCNK10 (TREK2)

1.4 References

Bardou O, Trinh NT, and Brochiero E. Molecular diversity and function of K+ channels in

airway and alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 296: L145-155, 2009.

Barfield JP, Yeung CH, and Cooper TG. The effects of putative K+ channel blockers on volume

regulation of murine spermatozoa. Biol Reprod 72: 1275-1281, 2005.

Bernard K, Bogliolo S, Soriani O, and Ehrenfeld J. Modulation of calcium-dependent chloride

secretion by basolateral SK4-like channels in a human bronchial cell line. J Membr Biol 196: 15-

31, 2003.

8

Chou K, Chen J, Yuan SX, and Haug A. The membrane potential changes polarity during

capacitation of murine epididymal sperm. Biochem Biophys Res Commun 165: 58-64, 1989.

Cowley EA and Linsdell P. Characterization of basolateral K+ channels underlying anion

secretion in the human airway cell line Calu-3. J Physiol 538: 747-757, 2002.

Doyle DA, Morais Cabral J, Pfuetzner RA, Kuo A, Gulbis JM, Cohen SL, Chait BT, and

MacKinnon R. The structure of the potassium channel: molecular basis of K+ conduction and

selectivity. Science 280: 69-77, 1998.

Dulong S, Bernard K, and Ehrenfeld J. Enhancement of P2Y6-induced Cl- secretion by IL-13

and modulation of SK4 channels activity in human bronchial cells. Cell Physiol Biochem 20:

483-494, 2007.

Duprat F, Lesage F, Fink M, Reyes R, Heurteaux C, and Lazdunski M. TASK, a human

background K+ channel to sense external pH variations near physiological pH. EMBO J 16:

5464-5471, 1997.

Gutman GA, Chandy KG, Adelman JP, Aiyar J, Bayliss DA, Clapham DE, Covarriubias M,

Desir GV, Furuichi K, Ganetzky B, Garcia ML, Grissmer S, Jan LY, Karschin A, Kim D,

Kuperschmidt S, Kurachi Y, Lazdunski M, Lesage F, Lester HA, McKinnon D, Nichols CG,

O'Kelly I, Robbins J, Robertson GA, Rudy B, Sanguinetti M, Seino S, Stuehmer W, Tamkun

MM, Vandenberg CA, Wei A, Wulff H, and Wymore RS. International Union of Pharmacology.

9

XLI. Compendium of voltage-gated ion channels: potassium channels. Pharmacol Rev 55: 583-

586, 2003.

Gutman GA, Chandy KG, Grissmer S, Lazdunski M, McKinnon D, Pardo LA, Robertson GA,

Rudy B, Sanguinetti MC, Stuhmer W, and Wang X. International Union of Pharmacology. LIII.

Nomenclature and molecular relationships of voltage-gated potassium channels. Pharmacol Rev

57: 473-508, 2005.

Hebert SC, Desir G, Giebisch G, and Wang W. Molecular diversity and regulation of renal

potassium channels. Physiol Rev 85: 319-371, 2005.

Heitzmann D and Warth R. Physiology and pathophysiology of potassium channels in

gastrointestinal epithelia. Physiol Rev 88: 1119-1182, 2008.

Jovanovic S, Crawford RM, Ranki HJ, and Jovanovic A. Large conductance Ca2+-activated K+

channels sense acute changes in oxygen tension in alveolar epithelial cells. Am J Respir Cell Mol

Biol 28: 363-372, 2003.

L'Hoste S, Poet M, Duranton C, Belfodil R, e Barriere H, Rubera I, Tauc M, Poujeol C, Barhanin

J, and Poujeol P. Role of TASK2 in the control of apoptotic volume decrease in proximal kidney

cells. J Biol Chem 282: 36692-36703, 2007.

10

Liu C, Cotten JF, Schuyler JA, Fahlman CS, Au JD, Bickler PE, and Yost CS. Protective effects

of TASK-3 (KCNK9) and related 2P K channels during cellular stress. Brain Res 1031: 164-173,

2005.

Lu Z. Mechanism of rectification in inward-rectifier K+ channels. Annu Rev Physiol 66: 103-

129, 2004.

Miller C. An overview of the potassium channel family. Genome Biol 1: REVIEWS0004, 2000.

O'Grady SM and Lee SY. Molecular diversity and function of voltage-gated (Kv) potassium

channels in epithelial cells. Int J Biochem Cell Biol 37: 1578-1594, 2005.

Patel AJ, Honore E, Lesage F, Fink M, Romey G, and Lazdunski M. Inhalational anesthetics

activate two-pore-domain background K+ channels. Nat Neurosci 2: 422-426, 1999.

Plaster NM, Tawil R, Tristani-Firouzi M, Canun S, Bendahhou S, Tsunoda A, Donaldson MR,

Iannaccone ST, Brunt E, Barohn R, Clark J, Deymeer F, George AL, Jr., Fish FA, Hahn A, Nitu

A, Ozdemir C, Serdaroglu P, Subramony SH, Wolfe G, Fu YH, and Ptacek LJ. Mutations in

Kir2.1 cause the developmental and episodic electrical phenotypes of Andersen's syndrome. Cell

105: 511-519, 2001.

11

Reyes R, Duprat F, Lesage F, Fink M, Salinas M, Farman N, and Lazdunski M. Cloning and

expression of a novel pH-sensitive two pore domain K+ channel from human kidney. J Biol

Chem 273: 30863-30869, 1998.

Schwab A. Ion channels and transporters on the move. News Physiol Sci 16: 29-33, 2001.

Simon DB and Lifton RP. The molecular basis of inherited hypokalemic alkalosis: Bartter's and

Gitelman's syndromes. Am J Physiol 271: F961-966, 1996.

Singh AK, Devor DC, Gerlach AC, Gondor M, Pilewski JM, and Bridges RJ. Stimulation of Cl-

secretion by chlorzoxazone. J Pharmacol Exp Ther 292: 778-787, 2000.

Warth R, Barriere H, Meneton P, Bloch M, Thomas J, Tauc M, Heitzmann D, Romeo E, Verrey

F, Mengual R, Guy N, Bendahhou S, Lesage F, Poujeol P, and Barhanin J. Proximal renal

tubular acidosis in TASK2 K+ channel-deficient mice reveals a mechanism for stabilizing

bicarbonate transport. Proc Natl Acad Sci U S A 101: 8215-8220, 2004

12

CHAPTER 2 - TASK-2 potassium channel supports ion secretion in

pig vas deferens epithelia

Pradeep R. Malreddy and Bruce D. Schultz

Department of Anatomy & Physiology, Kansas State University, Manhattan, KS 66506

This chapter has been submitted in this format for review to a peer reviewed journal.

13

2.1 Abstract

Epithelial cells lining the vas deferens modify the luminal contents to which sperm are

exposed in response to neuroendocrine, autocrine and lumicrine transmitters. The role and

identity of vas deferens epithelial potassium channels that provide the correct luminal

environment for sperm maturation and delivery have not yet been determined. Cultures of vas

deferens epithelial cells isolated from adult pigs were employed to investigate contributions of

selected ion channels to net flux. A two-pore potassium channel, TASK-2, was identified on the

apical membrane of cultured primary porcine vas deferens epithelial cells (1°PVD). Bupivacaine,

a known TASK-2 inhibitor, when added to the apical bathing solution, inhibited forskolin-

stimulated short circuit current, Isc, in a concentration dependent manner with a maximum

inhibition of 72 ± 6% and an IC50 of 7.4 ± 2.2 µM. Apical exposure of 1°PVD cells to quinidine,

lidocaine, and clofilium (other known TASK-2 blockers) inhibited forskolin-stimulated Isc in a

concentration dependent manner. Fitting a modified Michalis-Menten function to the data

revealed IC50 values of 274 µM, 531 µM, and 925 µM, respectively. Riluzole, a two-pore

potassium channel activator, stimulated bupivacaine-sensitive Isc, further confirming the

contribution of TASK-2 to net ion flux. Western blotting demonstrated the presence of TASK-2

immunoreactivity in 1°PVD cell lysates, while immunocytochemistry demonstrated apical

localization of the targeted epitope in virtually all cells lining native porcine vas deferens. These

results suggest that TASK-2 likely plays a role in vas deferens epithelial ion transport that may

account for the reportedly high concentration of potassium in the male reproductive duct lumen.

TASK-2 likely contributes to male fertility as an integral member of the regulated transport

processes that account for the luminal environment to which sperm are exposed.

14

2.2 Introduction

Vas deferens historically have been considered to be conduits for the transportation of

sperm and as a storage organ until ejaculation. Many studies have evaluated the fluid transport

across rete testis, efferent ducts and epididymis but none have evaluated the vas deferens (8, 20,

43). Nonetheless, the vas deferens functions to provide the correct luminal environment for

sperm maturation. Epithelium lining the vas deferens likely plays a major role in determining the

composition of the luminal fluids to which sperm are exposed. Initial studies in rat male

reproductive tract luminal fluid volume changes suggest the absorptive nature of the epithelia

lining the male reproductive tract with ~50% of reabsorption of fluids taking place between

seminiferous tubules and the caput epididymis and 50% of the remaining fluid reabsorption

between the caput and the vas deferens (26). More recent studies have shown that these epithelia

are capable of anion secretion in response to various neurotransmitters (4, 35), neuro-

hypophyseal hormones (18), and autacoids (33) at physiological concentrations. For electrogenic

anion secretion to occur, one or more basolateral K+ channels are likely required, along with

other ion transport mechanisms (37). However, there are reports demonstrating the presence of

apical K+ channels in certain anion-secreting epithelia including cells lining the epididymis (9),

lacrimal acinar cells (39), and Calu-3 cells, an epithelial cell line derived from human airway

(11). The concept of K+ channels on the apical membrane and their contribution in supporting

the anionic secretion is an emerging field.

The K+ concentration in the luminal fluid of vas deferens of rat (26), ram, boar, bull (10),

and hamster (40), and man (19) is high relative to the plasma concentration. This may be

15

attributable to three causes: 1) Selective absorption of water and other solutes in the seminiferous

tubules, epididymes (8), and perhaps vas deferens 2) Secretion of K+ by spermatozoa, and 3)

Secretion of K+ by epithelial cells lining the lumen of epididymis and vas deferens (38). Human

vas deferens epithelial cells reportedly have maxi K+ channel activity in the apical membranes

(38). Also, high K+ concentration in the rat caudal epididymal fluid has been shown to inhibit

initiation of sperm motility (42). Hence, it was speculated that K+ channels are located on the

apical plasma membrane of porcine vas deferens epithelial cells and could provide a pathway for

K+ secretion at this location. The luminal environment of vas deferens can be either acidic or

alkaline in nature depending upon the activity of proton and bicarbonate secreting mechanisms

(3, 5, 6). Thus, it seems logical that an acid-sensitive K+ channel might be present on the apical

membrane of the porcine vas deferens epithelial cells to account for regulated K+ secretion.

Two-pore K+ channels (K2P) are characterized by four transmembrane segments and two

pore domains as opposed to the usual one pore domains that are found in other K+ channel

families. The first member of the two-pore K+ channel family was named TWIK-1 for Tandem

of P domains in a Weak Inward Rectifying K+ channel (24, 25). Two-pore K+ channels have

been shown to be expressed in a number of different cell types since their discovery about twelve

years ago. Currently, fifteen mammalian genes have been identified that code for two-pore K+

channel family members. These channels have been grouped into different two-pore K+ channel

families based on their respective functional properties (17, 25). One of them is the TWIK-

related Acid-Sensitive K+ channel (TASK) family. The TASK channel family has five different

members. These channels are all sensitive to the extracellular pH (2, 12, 14, 21, 34) and exhibit

modest sequence homology (17). TASK-2 is a unique two-pore K+ channel with high sensitivity

16

to external pH in the physiological range and wide tissue distribution. TASK-2 mRNA is

expressed in various epithelial tissues including uterus, ovary, testis, kidney, lung, stomach,

intestine, colon, and salivary gland (34), but is poorly expressed or absent in excitable tissues

like brain and muscle (34). Whether TASK-2 or any other two-pore channel is present in porcine

vas deferens epithelial cells is not known.

The goal of this study was to determine whether TASK-2 K+ channels have a role in

modulating epithelial ion transport across porcine vas deferens epithelia. A two-pronged

approach was employed in which cultured cells were either placed in modified Ussing flux

chambers to test for effects on ion transport by known TASK-2 modulators or the cells were

lysed to probe protein samples with anti-TASK-2 antibodies. Finally, immunohistochemistry was

employed to provide evidence for the presence of TASK-2 on the apical membrane of epithelial

cells lining freshly isolated vas deferens.

2.3 Materials and Methods

2.3.1 Culture of Porcine Vas Deferens Epithelial Cells (1°PVD Cells)

Methods for isolation and culture of porcine vas deferens epithelial cells were reported in

detail previously (35). Briefly, adult male reproductive tracts were obtained from a local

slaughter house and maintained in ice-cold Ringer solution (composition in mM: 120 NaCl,

25 NaHCO3, 3.3 KH2PO4, 0.83 K2HPO4, 1.2 CaCl2, and 1.2 MgCl2) until further processing at

the laboratory. Those portions of the deferent ducts that extend from the transitional

epididymis/vas deferens to the prostate gland were used to isolate epithelial cells. Ducts were

17

flushed with a collagenase-based dissociation solution to obtain the epithelial cells that then were

seeded onto tissue culture flasks. Cells were incubated at 37°C at 5% CO2 in Dulbecco modified

Eagle medium (DMEM; GIBCO-BRL, Grand Island, NY) supplemented with 10% fetal bovine

serum (FBS; HyClone, Logan, UT) and 1% penicillin and streptomycin (Invitrogen, Carlsbad,

CA). The cells were allowed to grow to confluence (about 3-4 days) and then were subcultured

onto Snapwell permeable supports (1.13 cm2, Costar, Cambridge, MA). The media on both

basolateral and apical sides were changed the day following subculture and every other day

thereafter until assays were performed, typically 14 days after seeding.

2.3.2 Electrophysiology

Transepithelial electrical resistance (Rte), transepithelial potential difference (PDte), basal

short-circuit current (Isc) and changes in Isc stimulated/inhibited by select pharmacological agents

across 1°PVD monolayers on Snapwell permeable supports were assessed using a modified

Ussing chamber apparatus (Model DCV9, Navicyte, San Diego, CA). The experiments were

conducted in symmetrical Ringer solution at 39°C with continuous bubbling of 5% CO2 and 95%

O2. The monolayers were clamped to zero PDte using a voltage clamp apparatus (Model 558C,

University of Iowa, Department of Bioengineering, Iowa City, IA) and the resultant Isc recorded.

Monolayers were exposed to a 5 mV bipolar pulse of 5 s duration every 100 s and the

corresponding change in Isc was recorded. These parameters then were used to determine Rte

from Ohm's law, Rte = ΔV/ΔI. Data were acquired digitally at 1 Hz using an MP100A-CE

interface and Aqknowledge software (ver. 3.2.6, BIOPAC Systems, Santa Barbara, CA).

18

2.3.3 Immunohistochemistry

Small portions of porcine vas deferens were snap-frozen in liquid nitrogen and then

submerged in OCT embedding compound (Tissue-Tek, Sakura Finetek USA., Inc. Torrance,

CA). Sections of 10 or 12 µm thickness were cut using a Leica CM3050S Cryostat. The sections

were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) for 10

minutes at room temperature and rinsed with phosphate buffered saline for cytochemistry (PBS;

composition in mM: 5 KH2PO4, 15 K2HPO4, and 150 NaCl, pH 7.2-7.4). To prevent non-specific

binding, sections were incubated with 10% goat serum for 1 h at room temperature. Sections

were then incubated with anti-KCNK5 primary antibodies (Rabbit polyclonal, Alomone Labs,

Jerusalem, Israel) at 1:100 dilution in 2% normal goat serum overnight in a humid environment.

Secondary antibodies were prepared by diluting Alexa Fluor 488 goat anti-rabbit IgG (Molecular

Probes, Eugene, OR) in PBS containing 1% goat serum, and sections incubated for 45 minutes in

the dark at room temperature. The final incubation was with TO-PRO-3 iodide (Molecular

Probes) at 1:10,000 dilution for 20 minutes in the dark at room temperature. The slides were

mounted with Vectashield mounting medium for fluorescence (Vector Laboratories, Burlingame,

CA) and visualized using a Zeiss LSM 510 laser scanning confocal microscope.

2.3.4 1°PVD Whole Cell Lysate Preparation

Cell monolayers cultured on Snapwell permeable supports were used for whole cell

lysate preparation. The Snapwell trays containing cells were placed on ice; media removed by

aspiration and washed three times with PBS, with final wash being ice-cold PBS. RIPA buffer

19

(composition: 1 M TRIS-HCl pH 7.5, 1 M NaCl, 250 mM EDTA, 1% Triton-X, 1% sodium

deoxycholate, and 0.1% SDS) and protease inhibitor cocktail set III (Calbiochem, La Jolla, CA)

were mixed in a 1:100 ratio and added to each Snapwell permeable support (100 µL) to disrupt

the cell membranes. The cells were scraped using a pipette tip, aspirated using a 22 gauge

needle, transferred to a 1.5 mL eppendorf tubes and sheared by aspirating three times with a 26

gauge needle. The tubes containing lysate were placed on a rocker in a cold room for 2 hours.

The lysate was then spun at 15,000 rpm at 4°C for 5 minutes and the pellet was discarded. The

supernatant containing soluble protein was collected, transferred to new tubes and stored at -

20°C until further use. The protein concentrations in the lysates were measured using a

bicinchoninic acid assay kit (BCA; Pierce, Rockford, IL) just before performing Western blot

assays.

2.3.5 Pig Kidney Cortex Lysate Preparation

A small amount of pig kidney was obtained from a local slaughter house and the cortex

region was identified and immediately snap-frozen in liquid N2 until further processing at the

laboratory. About 1 g of frozen pig kidney cortex was powdered in liquid N2 using mortar and

pestle. 10 mL of RIPA buffer was added to the powdered tissue that was then homogenized

using a polytron with one burst of 20 s duration. The homogenized mixture was then centrifuged

at 15,000 rpm for 10 minutes at 4°C, supernatant collected and aliquoted into 100 µL and stored

at -80°C until used. The protein concentrations in the lysates were measured using BCA just

before performing Western blot assays.

20

2.3.6 Western Blotting

Protein samples were denatured by adding Laemmli sample buffer (Bio-Rad, Hercules,

CA) containing sodium dodecyl sulfate (SDS) and heating at 70°C for 10 minutes. β-

mercaptoethanol was added to create reducing conditions. Lysate samples (20 µg protein) were

loaded in each well of 4-20% SDS-polyacrylamide gel (Bio-Rad) and resolved by

electrophoresis with 30 mA for about 1 hour. Proteins were then transferred to polyvinylidene

fluoride (PVDF) transfer membrane (Millipore, Bedford, MA) under semi-dry conditions at 100

mA for 3 hours. Membranes were blocked overnight at 4°C in blocking buffer containing 5% fat

free dry milk (Nestle, Glendale, CA), and 0.025% sodium azide dissolved in PBS. The

membranes were then probed with anti-KCNK5 antibody or anti-KCNK5 antibody pre-adsorbed

with its immunizing peptide (Alomone, Jerusalem, Israel) in blocking buffer. Appropriate ECL

peroxidase labeled secondary antibody was used with a digital imaging system (Image Station

4000R, Kodak, Rochester, NY).

2.3.7 Chemicals

Penicillin, streptomycin, amphotericin-B, collagenase, gentamicin, and trypsin-EDTA

were purchased from Invitrogen (Carlsbad, CA). Forskolin was purchased from BioMol

(Plymouth Meeting, PA). Bupivacaine, lidocaine, quinidine, clofilium, and riluzole were all

purchased from Sigma-Aldrich (St. Louis, MO).

21

2.3.8 Data Analysis

Data are presented as means ± SE. Means were compared between treatments using a

Student's t-test. Statistical significance was assigned at P < 0.05.

2.4 Results

2.4.1 Apical Bupivacaine Inhibits Forskolin-Stimulated Isc in 1°PVD Cells

The cultured 1°PVD cells had basal Isc values near zero. Forskolin exposure was

employed to assess the responsiveness of the cell monolayers. The responses of 1°PVD cells to

forskolin were typical (35), which was characterized by an immediate peak in Isc followed by a

sustained plateau (see Fig. 2.1A). The increase in Isc was accompanied by a concurrent reduction

in Rte, suggesting that a conductive pathway is activated by forskolin. It has been demonstrated

previously with 1°PVD cells that an increase in Isc in response to forskolin is indicative of HCO3-

and/or Cl- secretion (5, 35). The sustained forskolin-stimulated Isc across 1°PVD cell monolayers

in current studies was 4.2 ± 0.1 µA/cm2 (n = 71). It was shown previously that pan-selective

potassium channel blockers reduced forskolin-stimulated Isc (35). Additional experiments are

required to determine the identity of channels that contribute to this response.

Bupivacaine, a local anesthetic, is known to inhibit two-pore potassium channels [TASK-

2 (23) and TREK-1 (25)] in both excitable and non-excitable cells. Results presented in Fig.

2.1A demonstrate that bupivacaine (100 _M), when added to the apical bathing solution,

inhibited the sustained component of Isc that was stimulated by forskolin. Basolateral exposure to

bupivacaine also reduced forskolin-stimulated Isc, but to a lesser extent and with slower kinetics.

22

Inhibition of forskolin-stimulated Isc by bupivacaine was concentration dependent (Fig. 2.1B). A

similar pattern is observed regardless whether one examines the absolute magnitude of inhibition

or the proportion of Isc that is inhibited. At the lowest concentration tested, 1 _M, bupivacaine

caused a small, but reproducible decrease in Isc. Incrementally greater inhibition was observed at

higher concentrations with an apparent plateau being reached at 100 to 300 _M. Proportional

inhibition was fitted by a modified Michalis-Menten equation, I = Imax*[x/(c+x)], where I is the

proportion of Isc that is inhibited, Imax is the derived maximal inhibition, x is concentration of

bupivacaine, and c is the concentration of bupivacaine causing a half-maximal inhibition (IC50).

The results indicate that bupivacaine inhibits a maximum of 71.6 ± 5.8% of forskolin-stimulated

Isc, while the IC50 was 7.4 ± 2.2 µM. The goodness of fit, as evaluated by eye and with a

modified Hill equation, suggests strongly that the data are well-described by a simple

bimolecular reaction scheme in which the interaction of a single bupivacaine molecule with a

single transport protein (e.g., channel) results in the immediate inhibition of all dependent

transport processes. Following exposure to bupivacaine, there was also a concentration-

dependent increase in Rte (Fig. 2.1C) that is consistent with the blockade of a potassium

conductive pathway. The dynamic portion of the sensitivity range to bupivacaine is similar to the

reported IC50 values for inhibition of TASK-2 channels by racemic bupivacaine (26 ± 5 µM

(22)), suggesting that TASK-2 inhibition by bupivacaine accounts for the reduction in Isc across

1°PVD monolayers.

23

Figure 2.1 Forskolin-stimulated Isc in primary porcine vas deferens epithelial cell

monolayers (1°PVD cells) is inhibited by apical application of bupivacaine.

A) Typical response of 1°PVD cells to forskolin (2 µM) and bupivacaine (100 µM apical). B) Summary

of concentration-dependent inhibition of change in forskolin-stimulated Isc by bupivacaine in 1°PVD cells

(bar graphs). Responses of 1°PVD cells to apical bupivacaine exposure are summarized as percent

inhibition of forskolin stimulated Isc. The solid line represents the best fit of a modified Michalis-Menten

equation to the data set (Fit parameters reported in the text). C) Summary of percent change in trans-

epithelial resistance of 1°PVD cells in response to the different concentrations of bupivacaine that were

tested. Each data point represents 3-5 observations, vertical bars indicate standard error of mean (SEM).

2.4.2 Lidocaine, Clofilium and Quinidine Inhibit Forskolin-Stimulated Isc across 1°PVD

Monolayers

We sought to determine whether other putative TASK-2 blockers, when applied to the

apical side of 1°PVD monolayers, had any effect on forskolin-stimulated Isc. Lidocaine, clofilium

and quinidine have been reported previously to inhibit TASK-2 and other two pore potassium

channels (7, 11, 22); hence experiments were conducted to determine the effects of these

pharmacological agents on forskolin-stimulated Isc across 1°PVD cell monolayers.

24

Apical application of lidocaine, similar to bupivacaine, inhibited sustained forskolin-

stimulated Isc across 1°PVD cells (Fig. 2.2A). However, basolateral application of lidocaine had

no effect. Inhibition by lidocaine was either incomplete or occurs at higher concentrations than

those required for bupivacaine as the subsequent apical exposure to an equal concentration of

bupivacaine (100 µM) resulted in substantially greater inhibition. Nonetheless, even over the

range that was tested, inhibition of forskolin-stimulated Isc was concentration dependent (Fig.

2.2D). These data were replotted as percent inhibition of the forskolin-stimulated Isc and fitted by

a modified Michalis-Menten equation that was constrained to a maximal inhibition of 71.6%

(equal to Imax derived for bupivacaine; Fig. 2.0.2E). The IC50 for inhibition of forskolin-

stimulated Isc by lidocaine was 531 ± 1834 µM. The concentration predicted to cause half-

maximal inhibition with this analysis is slightly less than was employed in other reports to

achieve TASK-2 inhibition (11, 22). Thus, the potency that is observed is in the anticipated range

based upon previous reports for the inhibition of TASK-2.

Clofilium reportedly inhibits potassium currents in mTASK-2 transfected HEK293 cells

with an IC50 of 25 µM (32), although it is also known to inhibit cAMP-gated potassium channels

in Calu-3 and human bronchial epithelial cells (11, 13). Apical clofilium exposure to 1°PVD

monolayers caused a modest decrease in forskolin-stimulated Isc (Fig. 2.2B) that, like lidocaine,

was superseded in magnitude by bupivacaine exposure. Inhibition by apical clofilium was clearly

concentration dependent (Fig. 2.2D), although, there was not a significant change in Isc at the

lowest concentration tested, 30 µM. Incrementally greater inhibition with 100 and 300 µM was

observed in paired studies. Fitting a modified Michalis-Menten function to the data (again, with

Imax constrained to 71.6%) revealed an extremely low potency of 925 ± 12800 µM. The

25

tremendous variation associated with this estimate is expected, given that less than 20%

inhibition was observed at the highest concentration tested, 300 µM. Basolateral exposure to

clofilium also inhibited Isc across 1°PVD cells, although with much slower kinetics indicating

that an alternative mechanism was likely affected in this approach. Rapid, concentration-

dependent inhibition by apical clofilium is consistent with targeting to apical TASK-2 channels,

although the concentrations required for inhibition are greater than expected. Thus, the effects of

a fourth putative TASK-2 antagonist were assessed.

Quinidine has been shown to inhibit potassium currents generated by TASK-2 transfected

HEK293 cells (34). Apical exposure of 1°PVD monolayers to quinidine, like bupivacaine,

lidocaine, and clofilium, inhibited forskolin-stimulated Isc in a concentration dependent manner

(Fig. 2.2D) while there was no effect of basolateral application. The IC50 for inhibition of

forskolin-stimulated Isc by quinidine when applied to the apical side was 274 ± 382 µM. Taken

together, results presented in Figs. 2.1 and 2.2 show concentration dependent inhibition of

forskolin-stimulated ion transport by four compounds that are reported to block TASK-2

channels with the following rank order of potency: bupivacaine>>quinidine>lidocaine>clofilium.

26

Figure 2.2 Forskolin-stimulated Isc across 1°PVD cells is inhibited by apical exposure to

lidocaine, clofilium or quinidine.

Typical response of 1°PVD cells to forskolin (2 µM) and A) lidocaine (100 µM apical) followed by

bupivacaine (100 µM apical), B) clofilium (100 µM apical) followed by bupivacaine, and C) quinidine

(100 µM apical). D) Summary of concentration-dependent inhibition of forskolin-stimulated Isc by

lidocaine, clofilium and quinidine across 1°PVD cell monolayers. E) Responses of 1oPVD cells to apical

lidocaine, clofilium and quinidine exposure are summarized as % inhibition of forskolin-stimulated Isc.

Solid or dashed lines represent the best fit of a modified Michalis-Menten equation to each data set.

Parameters of the fits are reported in the text. Data points in D and E represent 3-5 observations in each

condition and vertical bars indicate standard error of mean (SEM).

27

2.4.3 Riluzole, a two-pore potassium channel activator stimulates Isc across 1°PVD cells

Riluzole is a neuroprotective agent used in the treatment of amyotrophic lateral sclerosis

in humans that activates TREK-1 and TRAAK (15). Hence experiments were conducted to

determine if riluzole had any stimulatory effect on Isc across 1°PVD cells. Apical exposure to

riluzole (100 µM) caused a biphasic increase in Isc. There was a transient peak in Isc followed by

a sustained plateau (Fig. 2.3A). Basolateral application of riluzole also increased Isc; however the

response was slowly developing and small compared to the apical response (Fig. 2.3B), which

might result from passive diffusion across the monolayer to affect apical channels. Results

presented in Fig. 2.3C and D show that, in the presence of bupivacaine, the response to riluzole

is significantly less, an outcome expected if riluzole is activating TASK-2. Thus, the

pharmacology resulting from the use of five compounds, one activator and four blockers is

consistent with TASK-2 activity in the apical membrane being required to achieve maximal

anion secretion.

28

Figure 2.3 Apical application of riluzole stimulates Isc across 1°PVD cells.

Typical responses to A) apical or B) basolateral riluzole (100 µM) or C) apical riluzole in the presence of

bupivacaine are shown. Results are typical of 3-5 observations per condition. D) Summary of the peak

responses of 1°PVD cells to riluzole (100 µM apical) in the absence and presence of bupivacaine (100

µM apical). Results are summarized from 5 paired observations. Asterisk indicates a statistical significant

difference in the responses of 1°PVD cells to riluzole alone and riluzole plus bupivacaine.

2.4.4 1°PVD cells express TASK-2 immunoreactivity

Antibodies raised against amino acid residues 483-499 of human TASK-2 were used to

test for TASK-2 immunoreactivity in 1°PVD cell lysates. Two prominent bands were labeled

29

consistently at ~55 kDa by this antibody in the 1°PVD cell lysates (Fig. 2.4A) consistent with the

observations made by other researchers (11, 16), whereas only single band was labeled in pig

kidney cortex cell lysates (Fig. 2.4B), as expected. A band of greater mobility was also observed

consistently in the 1°PVD cell lysates (Fig. 2.4A). However, when the TASK-2 antibody was

pre-adsorbed with the immunizing peptide provided by the manufacturer, no labeling was

observed (Fig. 2.4C and D) indicating that the immunoreactivity was likely specific to TASK-2

expressed in 1°PVD cell and pig kidney cortex lysates.

A

55kDa

CB D

33kDa

Figure 2.4 TASK-2 immunoreactivity is present in 1°PVD cell lysates.

1°PVD (A & C) and pig kidney cortex (B & D) cell lysates were resolved by SDS-PAGE and probed with

antibodies raised against a TASK-2 epitope or the same antibodies that had been preadsorbed with the

immunizing peptide (C & D). Results are typical of three separate experiments.

2.4.5 Apical localization of TASK-2 protein in native porcine vas deferens ducts

Experiments were conducted to determine the localization of TASK-2 in freshly procured

porcine vas deferens. Tissues were processed and probed for TASK-2 immunoreactivity as

described in the METHODS. Images of anti-TASK-2 labeled and TO-PRO-3 iodide stained

sections of the porcine vas deferens duct were obtained as optical sections using a confocal

30

microscope (Fig. 2.5). A nearly continuous line of immunoreactivity is present at or near the

apical membrane of cells lining the duct, suggesting that the immunoreactive epitope is present

in the apical membrane of virtually all epithelial cells present in the sample. No labeling was

observed either when the primary antibody was omitted from the protocol or when the primary

antibody was pre-adsorbed with immunizing peptide (Fig. 2.5B & C). These results presented in

Figs. 2.4 and 2.5 provide strong evidence that TASK-2 is expressed by epithelial cells lining the

porcine vas deferens, which supports the observations employing pharmacological techniques to

suggest that TASK-2 at the apical epithelial membrane contributes to stimulated anion secretion.

A B

Lumen

C

Figure 2.5 TASK-2 immunoreactivity is present in cells lining native porcine vas deferens.

A) TASK-2 immunoreactivity (green) is localized to the apical region of virtually all cells lining native

porcine vas deferens. Immunolabeling is not observed when the primary antibody was omitted from the

assay protocol (B) and is virtually absent when the primary antibody was pre-adsorbed with immunizing

peptide (C). Nuclei are stained blue with TO-PRO-3 iodide. These results are typical of experiments

employing vas deferens derived from three different pigs.

2.5 Discussion

Evidence presented in this manuscript supports a cellular model to account for vas

deferens ion transport that includes a two-pore potassium channel, TASK-2, in the apical

membrane of epithelial cells lining the duct. A panel of pharmacological agents including four

31

compounds known to block TASK-2 channels and one compound that was expected to increase

TASK-2 activity had the expected effect to either reduce or enhance Isc, respectively. Inhibition

of Isc by each of the four blockers was concentration dependent and the rank order of potency is

consistent with the expectations for inhibition of TASK-2. Immunoreactivity for TASK-2 was

present in lysates derived from cultured vas deferens epithelial cells. Most importantly,

antibodies raised against TASK-2 provide a robust signal at the apical membrane of virtually all

epithelial cells lining the native duct. These results compel us to revise cell models that are

currently envisioned to account for the generation and/or modification of the reproductive duct

luminal contents by including an apical potassium conductance that contributes to the fluid and

solute environment to which sperm are exposed.

Models to account for epithelial anion secretion typically include channels that provide

an exit route for potassium, which enters the cell with the actions of the Na+/K+ ATPase and the

Na+/K+/2Cl- cotransporter. Potassium exit through membrane channels concurrently

hyperpolarizes the cell membrane, providing the electromotive force for anion exit at the apical

membrane. Such a cellular model was proposed for epithelial cells derived from porcine vas

deferens (35). Indeed, it was shown that basolateral Ba2+ and clotrimazole, but neither

charybdotoxin nor 293B, were associated with substantial inhibition of forskolin-stimulated Isc

(35). While the published model (35) accounts for anion secretion and the observed Isc, such a

model does not account for the possibility of potassium secretion, even though the potassium

concentration in luminal contents is reportedly greater than the concentration in plasma (10, 19,

26, 40). It was suggested some time ago that apical K+ channels might also support anion

secretion (9), and more recently it was reported that members of the two-pore potassium channel

32

family were expressed in the apical membrane of Calu-3 cells (11), a cell line derived from

human airway that has the characteristics of serous cells. Thus, experiments were designed and

conducted to test for the effects of two-pore K+ channel blockers on 1°PVD ion transport.

Bupivacaine, lidocaine, clofilium and quinidine, which are known to block TASK-2 (7,

11, 22, 23), reduced forskolin-stimulated Isc across 1°PVD monolayers. All of these compounds

inhibited forskolin-stimulated Isc in a concentration dependent manner when applied to the apical

membrane of 1°PVD monolayers. Following the addition of all these drugs, there was also a

concurrent concentration dependent increase in Rte that is consistent with the blockade of a

conductive pathway (i.e., a potassium channel). Bupivacaine was the most potent blocker with an

IC50 of <10 µM and the rank order of potency was bupivacaine>>quinidine>

lidocaine>clofilium. Only bupivacaine showed an apparent maximal effect in the concentration

range that was tested, inhibiting ~72% of forskolin-stimulated Isc. These blockers were most

effective when applied to the apical membrane of 1°PVD monolayers. Bupivacaine and clofilium

also showed inhibitory effects with basolateral exposure although the kinetics were slower and

the degree of inhibition appeared to be less, indicating that an alternative mechanism was

affected. It is possible that these agents are able to partition across the epithelial monolayer to

affect apical channels. Alternatively, it is possible that both an apical K+ channel (in this case,

TASK-2) and a basolateral bupivacaine- and clofilium-sensitive voltage gated K+ channel may

be activated for the electrogenic anion secretion to occur. Additionally riluzole, an activator of

two-pore potassium channels (15) stimulated an increase in Isc when applied to the apical side of

1°PVD cells. Taken together, these data strongly suggest that functional TASK-2 K+ channels

are present in the apical membrane of 1o PVD cells.

33

Western blotting revealed immunoreactivity of 1°PVD cell lysates to anti-TASK-2

antibodies and immunohistochemistry localized TASK-2 immunoreactivity to the apical side of

virtually all epithelial cells lining native porcine vas deferens. Taken together, these data suggest

that TASK-2 protein is present in porcine vas deferens epithelial cells and that it is functionally

active and expressed at the apical side of the epithelial cells lining the duct.

Unlike other two-pore potassium channels that are predominantly expressed in excitable

cells, TASK-2 is more prominently expressed in epithelial tissues such as in kidney, pancreas,

liver, small intestine, lung, placenta and uterus (28, 34). In fact, it is believed to be absent from

both human and mouse brain (1, 34, 41), in spite of few reports suggesting TASK-2 mRNA

expression in human dorsal root ganglia and spinal cord (29) and mouse taste receptor cells (27).

Nonetheless, TASK-2 has been increasingly gaining attention and is thought to play key roles in

epithelial ion transport processes such as cell volume regulation in primary cultures of mouse

proximal renal tubules (32), regulation of bicarbonate transport in mouse proximal renal tubules

(41), vectorial chloride transport in human pancreatic duct adenocarcinoma cell line (16), K+

exit/recycling across the apical membrane in Calu-3 cells (11), and in the maintenance of acid-

base homeostasis (31).

Initially it was surprising that apical exposure to TASK-2 blockers was associated with a

decrease in Isc because potassium secretion would generate a negative Isc and the inhibition of

cation secretion would necessarily increase Isc, opposite to what was observed. However, cells

derived from porcine vas deferens, like pancreatic duct cells, express a number of bicarbonate

34

transporters including SLC4A4 (sodium bicarbonate cotransporter), SLC26A3, SLC26A4,

SLC26A6 (chloride bicarbonate exchangers), and CFTR (chloride and bicarbonate permeant

channel) (5, 6). Importantly, both SLC26A3 and SLC26A6 (36) are reportedly electrogenic, as is

SLC4A4 (5). Thus, the possibility exists that the activation of TASK-2 supports bicarbonate

and/or chloride exits via electrogenic exchangers.

Ion transport across porcine vas deferens epithelial cells might be modeled as shown in

Fig. 2.6. CFTR and an electrogenic Cl-/HCO3- exchanger (e.g., SLC26A6) are present on the

apical membrane along with TASK-2. The basolateral membrane includes the Na+/K+ ATPase

that, in concert with an unidentified K+ conductance is thought to set the resting membrane

potential of the cell, a Na+/HCO3- cotransporter (SLC4A4), and NKCC, both of which harness

the electromotive force for Na+ to bring anions into the cell (5, 6, 35). It was proposed previously

that exposure to forskolin or to adrenergic or adenosine receptor agonists elicit an increase in Cl-

and HCO3- secretion (4, 5, 35). Exit of Cl- through CFTR would enhance the activity of the Cl-

/HCO3- exchanger. However, the activity of these two mechanisms, CFTR and SLC26A6 would

depolarize the membrane, which would limit anion secretion.

The presence of HCO3- in the lumen, however, would increase the activity of TASK-2,

which would repolarize the cell and maintain ongoing anion secretion. As long as there is a net

excess of Cl- and/or HCO3- exit across the apical membrane relative to the amount of K+ exit, the

activity of TASK-2 would tend to increase Isc. Alternatively the blockade of TASK-2 by

bupivacaine would depolarize the apical membrane, reduce exit of Cl- via CFTR and reduce the

driving force for the electrogenic anion exchanger, which would be seen as a decrease in Isc.

35

Na+

HCO3-

2HCO3-

Na+K+2Cl-

K+

K+K+

Na+

Cl-

Cl-

Apical Basolateral

BupivacaineTASK -2

SLC26A6

CFTR

+

NKCC

NBC

Na+ Pump

Na+

HCO3-

Na+

HCO3-

2HCO3-

Na+K+2Cl-

Na+K+2Cl-

K+K+

K+K+

Na+

K+

Na+

Cl-

Cl-

Apical Basolateral

BupivacaineTASK -2

SLC26A6

CFTR

+

NKCC

NBC

Na+ Pump

Figure 2.6 Porcine vas deferens epithelial cell model for ion transport.

It has been suggested that TASK-2 acts as a molecular switch for K+ conductance and

bicarbonate transport activity (41). In this proposed scheme, an increase in extra-cellular pH due

to HCO3- secretion activates the pH-sensitive TASK-2 to permit K+ exit and thereby

hyperpolarizes or repolarizes the cell membrane. This scenario can be used to explain functional

significance of basolateral localization of TASK-2 in proximal renal tubule cells where there is

HCO3- reabsorption (41) and the apical localization of TASK-2 in pancreatic (16) and lung

epithelial cell lines (11) where there is HCO3- secretion. 1°PVD cells and PVD9902 cells, an

epithelial cell line derived from normal porcine vas deferens, are capable of HCO3- secretion and

the results from the current study provide functional and molecular evidence for TASK-2

expression in the apical membrane of primary cultured and native porcine vas deferens epithelia,

36

suggesting that such a mechanism for HCO3- associated K+ secretion may be present in this

tissue.

This positive feedback loop that includes TASK-2 support for HCO3- secretion can be

envisioned to have a physiological role in male fertility. Sperm are quiescent in an acid

environment, which has been reported for the epididymis and vas deferens, and the sperm

become active in an alkaline environment. Thus, during emotional stimulation prior to

ejaculation, norepinephrine released from the hypogastric nerve would be expected to stimulate

Cl- and HCO3- secretion across vas deferens and perhaps distal epididymal epithelium. The

secreted HCO3- would activate TASK-2 to support ongoing HCO3

- secretion. Sperm in this

region of the duct would initiate activity due to HCO3- exposure. At ejaculation, the alkaline

contents of the lumen would be propelled forward and the more acid contents from proximal

regions of the epididymis would be propelled into this region of the duct to cause an immediate

reduction in TASK-2 activity. Concurrently, norepinephrine release from the hypogastric nerve

would cease and the epithelial cells would return to basal activity that includes little or no HCO3-

secretion. Any sperm in this region of the duct would be stored in a quiescent state until HCO3-

secretion is again stimulated. Thus, TASK-2 contributes to a system that modifies the luminal

environment to either store sperm in a quiescent state or to activate sperm during arousal.

In conclusion, this report suggests that TASK-2 is present in the apical membrane of

1oPVD cells and may contribute to greater K+ concentration in vas luminal fluids that may be

required for maturation of sperm to occur.

37

2.6 Acknowledgements

The authors would like to thank Mr. Ryan Carlin and Drs. Rebecca Quesnell and Suma

Somasekharan for technical assistance. Appreciation is also expressed to Henry’s Ltd, Dr.

Fernando Pierucci-Alves and Florence Wang for their assistance with tissue procurement. This

project utilized the KSU-COBRE Center for Epithelial Function in Health and Disease’s

Confocal Microscopy and Molecular Biology Core Facilities.

2.7 References

1. Aller MI, Veale EL, Linden AM, Sandu C, Schwaninger M, Evans LJ, Korpi ER,

Mathie A, Wisden W, and Brickley SG. Modifying the subunit composition of TASK channels

alters the modulation of a leak conductance in cerebellar granule neurons. J Neurosci 25: 11455-

11467, 2005.

2. Ashmole I, Goodwin PA, and Stanfield PR. TASK-5, a novel member of the tandem

pore K+ channel family. Pflugers Arch 442: 828-833, 2001.

3. Brown D and Breton S. H+V-ATPase-dependent luminal acidification in the kidney

collecting duct and the epididymis/vas deferens: vesicle recycling and transcytotic pathways. J

Exp Biol 203: 137-145, 2000.

38

4. Carlin RW, Lee JH, Marcus DC, and Schultz BD. Adenosine stimulates anion

secretion across cultured and native adult human vas deferens epithelia. Biol Reprod 68: 1027-

1034, 2003.

5. Carlin RW, Quesnell RR, Zheng L, Mitchell KE, and Schultz BD. Functional and

molecular evidence for Na+-HCO3- cotransporter in porcine vas deferens epithelia. Am J Physiol

Cell Physiol 283: C1033-1044, 2002.

6. Carlin RW, Sedlacek RL, Quesnell RR, Pierucci-Alves F, Grieger DM, and Schultz

BD. PVD9902, a porcine vas deferens epithelial cell line that exhibits neurotransmitter-

stimulated anion secretion and expresses numerous HCO3- transporters. Am J Physiol Cell

Physiol 290: C1560-1571, 2006.

7. Cho SY, Beckett EA, Baker SA, Han I, Park KJ, Monaghan K, Ward SM, Sanders

KM, and Koh SD. A pH-sensitive potassium conductance (TASK) and its function in the

murine gastrointestinal tract. J Physiol 565: 243-259, 2005.

8. Clulow J, Jones RC, and Hansen LA. Micropuncture and cannulation studies of fluid

composition and transport in the ductuli efferentes testis of the rat: comparisons with the

homologous metanephric proximal tubule. Exp Physiol 79: 915-928, 1994.

9. Cook DI and Young JA. Effect of K+ channels in the apical plasma membrane on

epithelial secretion based on secondary active Cl- transport. J Membr Biol 110: 139-146, 1989.

39

10. Crabo B. Studies on the Composition of Epididymal Content in Bulls and Boars. Acta

Vet Scand 22: SUPPL 5:1-94, 1965.

11. Davis KA and Cowley EA. Two-pore-domain potassium channels support anion

secretion from human airway Calu-3 epithelial cells. Pflugers Arch 451: 631-641, 2006.

12. Decher N, Maier M, Dittrich W, Gassenhuber J, Bruggemann A, Busch AE, and

Steinmeyer K. Characterization of TASK-4, a novel member of the pH-sensitive, two-pore

domain potassium channel family. FEBS Lett 492: 84-89, 2001.

13. Devor DC, Bridges RJ, and Pilewski JM. Pharmacological modulation of ion transport

across wild-type and DeltaF508 CFTR-expressing human bronchial epithelia. Am J Physiol Cell

Physiol 279: C461-479, 2000.

14. Duprat F, Lesage F, Fink M, Reyes R, Heurteaux C, and Lazdunski M. TASK, a

human background K+ channel to sense external pH variations near physiological pH. EMBO J

16: 5464-5471, 1997.

15. Duprat F, Lesage F, Patel AJ, Fink M, Romey G, and Lazdunski M. The

neuroprotective agent riluzole activates the two P domain K+ channels TREK-1 and TRAAK.

Mol Pharmacol 57: 906-912, 2000.

40

16. Fong P, Argent BE, Guggino WB, and Gray MA. Characterization of vectorial

chloride transport pathways in the human pancreatic duct adenocarcinoma cell line HPAF. Am J

Physiol Cell Physiol 285: C433-445, 2003.

17. Goldstein SA, Bayliss DA, Kim D, Lesage F, Plant LD, and Rajan S. International

Union of Pharmacology. LV. Nomenclature and molecular relationships of two-P potassium

channels. Pharmacol Rev 57: 527-540, 2005.

18. Hagedorn TM, Carlin RW, and Schultz BD. Oxytocin and vasopressin stimulate anion

secretion by human and porcine vas deferens epithelia. Biol Reprod 77: 416-424, 2007.

19. Hinton BT, Pryor JP, Hirsh AV, and Setchell BP. The concentration of some

inorganic ions and organic compounds in the luminal fluid of the human ductus deferens. Int J

Androl 4: 457-461, 1981.

20. Jones RC. Luminal composition and maturation of spermatozoa in the genital ducts of

the African elephant (Loxodonta africana). J Reprod Fertil 60: 87-93, 1980.

21. Kim Y, Bang H, and Kim D. TASK-3, a new member of the tandem pore K+ channel

family. J Biol Chem 275: 9340-9347, 2000.

41

22. Kindler CH, Paul M, Zou H, Liu C, Winegar BD, Gray AT, and Yost CS. Amide

local anesthetics potently inhibit the human tandem pore domain background K+ channel TASK-

2 (KCNK5). J Pharmacol Exp Ther 306: 84-92, 2003.

23. Kindler CH, Yost CS, and Gray AT. Local anesthetic inhibition of baseline potassium

channels with two pore domains in tandem. Anesthesiology 90: 1092-1102, 1999.

24. Lesage F, Guillemare E, Fink M, Duprat F, Lazdunski M, Romey G, and Barhanin

J. TWIK-1, a ubiquitous human weakly inward rectifying K+ channel with a novel structure.

EMBO J 15: 1004-1011, 1996.

25. Lesage F and Lazdunski M. Molecular and functional properties of two-pore-domain

potassium channels. Am J Physiol Renal Physiol 279: F793-801, 2000.

26. Levine N and Marsh DJ. Micropuncture studies of the electrochemical aspects of fluid

and electrolyte transport in individual seminiferous tubules, the epididymis and the vas deferens

in rats. J Physiol 213: 557-570, 1971.

27. Lin W, Burks CA, Hansen DR, Kinnamon SC, and Gilbertson TA. Taste receptor

cells express pH-sensitive leak K+ channels. J Neurophysiol 92: 2909-2919, 2004.

28. Lotshaw DP. Biophysical, pharmacological, and functional characteristics of cloned and

native mammalian two-pore domain K+ channels. Cell Biochem Biophys 47: 209-256, 2007.

42

29. Medhurst AD, Rennie G, Chapman CG, Meadows H, Duckworth MD, Kelsell RE,

Gloger, II, and Pangalos MN. Distribution analysis of human two pore domain potassium

channels in tissues of the central nervous system and periphery. Brain Res Mol Brain Res 86:

101-114, 2001.

30. Miller C. An overview of the potassium channel family. Genome Biol 1:

REVIEWS0004, 2000.

31. Morton MJ, Abohamed A, Sivaprasadarao A, and Hunter M. pH sensing in the two-

pore domain K+ channel, TASK2. Proc Natl Acad Sci U S A 102: 16102-16106, 2005.

32. Niemeyer MI, Cid LP, Barros LF, and Sepulveda FV. Modulation of the two-pore

domain acid-sensitive K+ channel TASK-2 (KCNK5) by changes in cell volume. J Biol Chem

276: 43166-43174, 2001.

33. Pierucci-Alves F and Schultz BD. Bradykinin-stimulated cyclooxygenase activity

stimulates vas deferens epithelial anion secretion in vitro in swine and humans. Biol Reprod 79:

501-509, 2008.

34. Reyes R, Duprat F, Lesage F, Fink M, Salinas M, Farman N, and Lazdunski M.

Cloning and expression of a novel pH-sensitive two pore domain K+ channel from human

kidney. J Biol Chem 273: 30863-30869, 1998.

43

35. Sedlacek RL, Carlin RW, Singh AK, and Schultz BD. Neurotransmitter-stimulated ion

transport by cultured porcine vas deferens epithelium. Am J Physiol Renal Physiol 281: F557-

570, 2001.

36. Shcheynikov N, Yang D, Wang Y, Zeng W, Karniski LP, So I, Wall SM, and

Muallem S. The Slc26a4 transporter functions as an electroneutral Cl-/I-/HCO3- exchanger: role

of Slc26a4 and Slc26a6 in I- and HCO3- secretion and in regulation of CFTR in the parotid duct.

J Physiol 586: 3813-3824, 2008.

37. Silva P, Stoff J, Field M, Fine L, Forrest JN, and Epstein FH. Mechanism of active

chloride secretion by shark rectal gland: role of Na+-K+-ATPase in chloride transport. Am J

Physiol 233: F298-306, 1977.

38. Sohma Y, Harris A, Wardle CJ, Gray MA, and Argent BE. Maxi K+ channels on

human vas deferens epithelial cells. J Membr Biol 141: 69-82, 1994.

39. Tan YP and Young JA. Effect of K+ channels in the luminal membrane of lacrimal

acinar cells. J Membr Biol 89: 139-146, 1989.

40. Turner TT, Hartmann PK, and Howards SS. In vivo sodium, potassium, and sperm

concentrations in the rat epididymis. Fertil Steril 28: 191-194, 1977.

44

41. Warth R, Barriere H, Meneton P, Bloch M, Thomas J, Tauc M, Heitzmann D,

Romeo E, Verrey F, Mengual R, Guy N, Bendahhou S, Lesage F, Poujeol P, and Barhanin

J. Proximal renal tubular acidosis in TASK2 K+ channel-deficient mice reveals a mechanism for

stabilizing bicarbonate transport. Proc Natl Acad Sci U S A 101: 8215-8220, 2004.

42. Wong PY and Lee WM. Potassium movement during sodium-induced motility initiation

in the rat caudal epididymal spermatozoa. Biol Reprod 28: 206-212, 1983.

43. Wong PY and Yeung CH. Absorptive and secretory functions of the perfused rat cauda

epididymidis. J Physiol 275: 13-26, 1978.

45

CHAPTER 3 - Potential experiments and future directions

3.1 Overview

The results presented in Chapter 2 certainly support the contention that TASK-2 is

present in the apical membrane of epithelial cells lining porcine vas deferens. However, these

results are not unequivocal. None of the pharmacological agents employed are specific (or even

highly selective) for TASK-2. Thus one or more alternative K+ channels may have been targeted

by these agents over the concentration ranges tested. For example, 100 µM quinidine has been

reported to inhibit TWIK-1 and TREK-1 in addition to TASK-2. Also, the IC50 for inhibition of

voltage-gated Na+ channels in HEK293 cells by bupivacaine is in the same range (4.5 µM; Wang

et al, 2001) as seen in our inhibition of forskolin-stimulated Isc by bupivacaine in porcine vas

deferens epithelial cells (7.4 µM, see Results, Chapter 2). Likewise, Western blots and

immunohistochemistry provide evidence that the epitope derived from TASK-2 is present at the

apical membrane of these epithelial cells. However, there is the possibility that this epitope is

present in another protein and there is the possibility that protein carrying this epitope, regardless

of its identity, is not integrated into the apical membrane. Thus, additional lines of evidence

could be generated to test the hypothesis that TASK-2 is present in porcine vas deferens

epithelial cell apical membranes and contributes to net anion secretion.

46

Gene microarray of RNA isolated from porcine vas deferens epithelial cells can be used

to examine the expression of various K+ channel genes. Such K+ channel candidates can then be

selected for subsequent RT-PCR analyses to confirm the presence of full length mRNA

transcripts and to determine relative abundance of each coding message. Presence of mRNA

however, does not confirm the presence of proteins or that those gene products contribute to net

ion transport. Rather, the presence of mRNA provides evidence that the message is in place to

synthesize TASK-2 or other K2P channels.

Immunoreactivity is employed to implicate the expression of an epitope that is unique to

a protein of interest. Indeed, results presented in Fig. 2.4 demonstrate that a protein of the

expected mobility is labeled by an antibody raised against a TASK-2 epitope. To bolster the

veracity of these data, additional Western blotting can be performed using antibodies that are

raised against epitopes derived from both N- and C-termini of TASK-2. Lysates from porcine

kidney cortex can be analyzed in parallel since TASK-2 expression has been reported routinely

for the kidney. The kidney lysate samples would be expected to reveal a single band of expected

mobility with each antibody for comparison to the vas deferens cell lysates (as shown in Fig.

2.4). To confirm the identity of the TASK-2 protein in these cells, the porcine vas deferens

epithelial cell lysate can be immunoprecipitated with anti-TASK-2 antibody, resolved by SDS-

PAGE, visualized by Coomassie blue, excised at the expected mobility and analyzed using

Matrix-Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) mass spectrometry

to confirm the protein identity. Results from these experiments will provide an unequivocal line

of evidence for TASK-2 protein expression in porcine vas deferens epithelial cells.

47

Whether this TASK-2 protein is located at apical, basolateral or both surfaces is of

crucial physiological importance in porcine vas deferens epithelial cells. In order to determine

this localization, immunofluorescence using confocal microscopy was employed, which revealed

labeling by anti-TASK-2 antibodies of virtually all the cells lining the lumen of a freshly frozen

porcine vas deferens (Fig. 2.5). However, it can not be ascertained from this technique whether

the immunolabeling observed is in the apical cell membrane, or in the sub-apical vesicles of the

cells lining the lumen of vas deferens. Apical membrane incorporation can be verified by

performing cell-surface biotinylation wherein biotin is conjugated to the apical or basolateral cell

surface proteins without altering their biological activity and then detecting that labeled protein

by Western blotting with anti-TASK-2 antibody using avidin.

Functional studies can be conducted to test for TASK-2 activity in the apical membrane

of the porcine vas deferens epithelial cells. TASK-2 channels are sensitive to changes in

extracellular pH with maximal activity reported at pH 8.8 and progressively less current as pH

was decreased (Warth et al, 2004). In the current context, forskolin-stimulated Isc can be

measured in 1°PVD cells that are maintained in normal physiological salt solutions that are

buffered to selected pH values ranging from 5.4 to 9.4. The experiment can be conducted in two

different ways. One way is to measure the magnitude of forskolin-stimulated Isc in 1°PVD cells

that are bathed on the basolateral side with HEPES buffered saline (pH 7.4) and on the apical

side with different buffer solutions having pH values of 5.4 (citrate buffer), 6.4 (MOPS buffer),

7.4 (HEPES buffer), 8.4 (Tris buffer), and 9.4 (borate buffer). If TASK-2 is rate limiting for ion

transport, the expectation is that the greatest response to forskolin will be observed in the

chamber that is exposed to apical pH of 8.4 or 9.4. A further test of this hypothesis would

48

include apical exposure to bupivacaine (100 µM) with the expectation that the magnitude of the

bupivacaine-sensitive current would be greatest at pH 8.4 or 9.4 and that there would be no

bupivacaine-sensitive current at pH 5.4. An alternative, although more difficult, approach to test

for pH sensitivity in the response to forskolin is to place the monolayers at symmetrical pH of

7.4, expose the cells to forskolin to maximize Isc, and then adjust the apical pH either up or down

in the absence or presence of apical bupivacaine. If TASK-2 is involved in the response, the

expectation would be that the magnitude of Isc would increase as pH was increased and decrease

as the pH was lowered, but only in the monolayers not exposed to bupivacaine. While there may

be other concerns regarding the practical implementation of these experiments that must be

addressed concurrently (e.g., is pH 5.4 or 9.4 detrimental to the cell monolayers), the outcomes

will provide strong evidence to either exclude or incorporate TASK-2 in the epithelial apical

membrane as depicted in Fig. 2.6.

An alternative method to implicate the presence of TASK-2 in the apical membrane of

1°PVD cells is to test for bupivacaine-sensitive K+-dependent Isc across isolated apical

membranes. These experiments are conducted by establishing an apical to basolateral gradient in

the concentration of K+ (replace apical Na+ with K+) and permeabilizing the basolateral

membrane with nystatin. TASK-2 activity could be maximized by employing an apical pH of 8.4

and exposing the monolayer to forskolin. If TASK-2 is present and active in the apical

membrane, the Isc should be positive, indicating net cation absorption and the Isc should be

reduced substantially by bupivacaine. Since there is no gradient for Cl- secretion in this

arrangement and since the Na+ concentration gradient would tend to decrease Isc, the expected

results would rule out the possibilities that bupivacaine is having the affect on intact tissues by

49

inhibiting either Na+ or Cl- channels. The expected outcome would support the conclusion that

TASK-2 is actually integrated into the apical membrane and contributes to the observed Isc.

So, are TASK-2 channels required to observe a maximal Isc response to forskolin? One

way to answer this question is by reducing or eliminating TASK-2 expression using RNA

interference (RNAi). It has been reported that TASK-2 protein expression and current were

significantly down regulated in TASK-2 stably transfected HEK293 cells following the

incorporation of small interfering RNA (siRNA) against TASK-2 (Gonczi et al, 2006). The

effectiveness of RNAi can be determined at the mRNA and protein levels by RT-PCR and

Western blotting, respectively. Any difference in the magnitude of forskolin-stimulated Isc

response between control 1°PVD cells and 1°PVD cells transfected with siRNA against TASK-2

can be suggestive of a role for TASK-2 channels in anion secretion. In 1°PVD cells treated with

TASK-2 siRNA, forskolin would be expected to stimulate some, albeit reduced, level of Isc.

However, this response would be expected to be insensitive to either changes in apical pH or to

bupivacaine. Such an outcome would provide the most compelling evidence that TASK-2 is

required for maximal ion transport across vas deferens epithelial cells.

In conclusion, a cellular model that incorporates TASK-2 in the apical membrane is

presented in Fig. 2.6 and evidence supporting this model is presented throughout chapter 2. Each

figure provided evidence that was supportive of the model, but in isolation was not compelling.

The combination of techniques, however, provides a body of evidence that is substantive, but

neither exhaustive nor definitive. Thus, additional techniques and reagents as outlined in the

current chapter can be used to test the model presented in Fig. 2.6 further. Each of the suggested

50

experiments tests a distinct hypothesis regarding the expression and function of TASK-2. Some

or all of these techniques will likely be required to provide evidence that is viewed as compelling

by many scientists in this field.

3.2 References

Gonczi M, Szentandrassy N, Johnson IT, Heagerty AM, and Weston AH. Investigation of the

role of TASK-2 channels in rat pulmonary arteries; pharmacological and functional

studies following RNA interference procedures. Br J Pharmacol 147: 496-505, 2006.

Warth R, Barriere H, Meneton P, Bloch M, Thomas J, Tauc M, Heitzmann D, Romeo E, Verrey

F, Mengual R, Guy N, Bendahhou S, Lesage F, Poujeol P, and Barhanin J. Proximal renal

tubular acidosis in TASK2 K+ channel-deficient mice reveals a mechanism for stabilizing

bicarbonate transport. Proc Natl Acad Sci U S A 101: 8215-8220, 2004.